1. Field of the Invention
The present invention relates to methods and reagents for detecting the presence of nucleic acid from human strains of Pneumocystis carinii (P. carinii).
2. Description of Related Art
P. carinii is an opportunistic fungal pathogen that infects the lower respiratory tract. The fungal cysts and trophozoites cause severe pneumonia in humans immunocompromised as a result of AIDS, organ transplantation, bone marrow transplantation; or chemotherapy. P. carinii is the leading cause of morbidity and mortality in AIDS patients in the United States. Early diagnosis and treatment is essential to prevent mortality. Information relating to its taxonomic classification and characteristics can be found in Edman et al., 1988, Nature 334:519-522, incorporated herein by reference.
Initial methods for the diagnosis of P. carinii pneumonia (PCP) depended on the staining and microscopic examination of induced sputum, bronchial alveolar lavage (BAL) and transbronchial biopsy (TBB) specimens for evidence of the parasite. If PCP is suspected but an initial examination of an induced sputum sample is negative, the more invasive procedures of examining BAL or TBB specimens are required for diagnosis.
The invention of the polymerase chain reaction (PCR), a method for amplifying specific sequences of nucleic acids, makes possible the rapid detection of nucleic acids present in a cell in what was previously an undetectably low quantity (see U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein by reference). Using PCR amplification, one can detect even a single copy of the target nucleic acid. Direct detection of an amplified nucleic acid sequence by hybridization with a sequence-specific oligonucleotide probe makes possible diagnostic tests that are specific enough to detect single nucleotide changes in sequence.
Recent attempts have been made to develop a diagnostic test for P. carinii using nucleic acid amplification techniques. Edman et al., 1988, supra disclose a sequence of a region of the 16S-like (18S) ribosomal RNA (rRNA) gene of a P. carinii strain derived from rats. PCT publication WO 90/13669, incorporated herein by reference, discloses detection probes based on the rat-derived sequence published in Edman et al., 1988, supra. Lipschik, 1992, Lancet 340:203-206, incorporated herein by reference, also discloses primers and probes based on the sequence disclosed in Edman et al., 1988, supra, and describes their use in a nested PCR.
PCF publication WO 91/02092 describes the use of nonspecific amplification primers from Merlin et at., 1988, Gene 71:491-499, both of which are incorporated herein by reference, and probes based on the sequence of the 18S rRNA gene from a human strain of P. carinii. The amplification primers used to amplify a region of the 18S rRNA gene were not specific to P. carinii. The detection probes described typically hybridized to several of the human, rat, and ferret strains of P. carinii tested.
Detection assays based on gene sequences obtained from rat strains of P. carinii may be less sensitive and less specific than one which could be designed based on gene sequences obtained from a human strain of P. carinii because of genotypic variation between rat and human isolates of P. carinii. Thus, it would be advantageous to provide assays comprising primers and probes based on gene sequences obtained from a human strain of P. carinii. Furthermore, because it is unknown if rat strains of P. carinii can infect humans, it is desirable that an assay be able to distinguish between rat and human strains of P. carinii. There is still a need for a rapid, efficient, and sensitive assay to identify the presence of P. carinii DNA which is specific for human strains of P. carinii.